Anticancer compounds and methods

ABSTRACT

The testing of tumor cells, including human tumors capable of metastases, in assays employing fibronectin-depleted substrates is described. Ex vivo induction of cells, including biopsied human cells, is performed with invasion-inducing agents. Additionally, anti-cancer chemotherapeutics are described. Specifically, chemotherapeutic agents which have anti-metastatic and anti-growth properties are described including non-peptide compositions of matter.

[0001] This is a Continuation application of Ser. No. 08/915,189 filedon Aug. 20, 1997 which is a Continuation-In-Part of Ser. No. 08/754,322which subsequently issued as U.S. Pat. No. 5,840,514 on Nov. 24, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates to the treatment of cancer, to thetesting of cancer cells for their ability to invade tissues and causemetastases, and to the identification and use of drugs to inhibit tumorinvasion and growth.

BACKGROUND

[0003] The term “chemotherapy” simply means the treatment of diseasewith chemical substances. The father of chemotherapy, Paul Ehrlich,imagined the perfect chemotherapeutic as a “magic bullet”; such acompound would kill invading organisms without harming the host. Thistarget specificity is sought in all types of chemotherapeutics,including anticancer agents.

[0004] However, specificity has been the major problem with anticanceragents. In the case of anticancer agents, the drug needs to distinguishbetween host cells that are cancerous and host cells that are notcancerous. The vast bulk of anticancer drugs are indiscriminate at thislevel. Typically anticancer agents have negative hematological effects(e.g., cessation of mitosis and disintegration of formed elements inmarrow and lymphoid tissues), and immunosuppressive action (e.g.,depressed cell counts), as well as a severe impact on epithelial tissues(e.g., intestinal mucosa), reproductive tissues (e.g., impairment ofspermatogenesis), and the nervous system. P. Calabresi and B. A.Chabner, In: Goodman and Gilman The Pharmacological Basis ofTherapeutics (Pergamon Press, 8th Edition) (pp. 1209-1216).

[0005] Success with chemotherapeutics as anticancer agents has also beenhampered by the phenomenon of multiple drug resistance, resistance to awide range of structurally unrelated cytotoxic anticancer compounds. J.H. Gerlach et al., Cancer Surveys, 5:25-46 (1986). The underlying causeof progressive drug resistance may be due to a small population ofdrug-resistant cells within the tumor (e.g., mutant cells) at the timeof diagnosis. J. H. Goldie and Andrew J. Coldman, Cancer Research,44:3643-3653 (1984). Treating such a tumor with a single drug firstresults in a remission, where the tumor shrinks in size as a result ofthe killing of the predominant drug-sensitive cells. With thedrug-sensitive cells gone, the remaining drug-resistant cells continueto multiply and eventually dominate the cell population of the tumor.

[0006] Finally, the treatment of cancer has been hampered by the factthat there is considerable heterogeneity even within one type of cancer.Some cancers, for example, have the ability to invade tissues anddisplay an aggressive course of growth characterized by metastases.These tumors generally are associated with a poor outcome for thepatient. And yet, without a means of identifying such tumors anddistinguishing such tumors from non-invasive cancer, the physician is ata loss to change and/or optimize therapy.

[0007] What is needed is a specific anticancer approach that is reliablefor a wide variety of tumor types, and particularly suitable forinvasive tumors. Importantly, the treatment must be effective withminimal host toxicity.

SUMMARY OF THE INVENTION

[0008] The present invention relates to the treatment of cancer, to thetesting of cancer cells for their ability to invade tissues and causemetastases, and to the identification and use of drugs to inhibit tumorinvasion and growth. The present invention provides: A) an in vitromodel for testing cancer cells and evaluating invasive potential; B) ascreening assay for identifying drugs that inhibit tumor invasion; andC) chemotherapeutics for treating cancer.

[0009] A variety of assay formats are contemplated for testing theinvasive potential of cancer cells. In one embodiment, a portion of apatient's tumor is obtained (e.g., by biopsy) and placed in tissueculture on a fibronectin-free substrate. Thereafter, the response of thetumor cells to fibronectin or a fibronectin-derived peptide is assessed.Where fibronectin induces invasion of the membrane, the tumor can beconsidered to have metastatic potential. Where there is no significantinvasion of the membrane, the tumor can be considered (at that time) tobe non-metastatic.

[0010] In one embodiment, the present invention contemplates a method ofevaluating human cancer comprising: a) providing: i) a human cancerpatient, ii) a fibronectin-free substrate, and iii) one or moreinvasion-inducing agents; b) obtaining cancer cells from said patient;c) contacting said cells ex vivo with said fibronectin-free substrateand one or more invasion-inducing agents; and d) detecting cancer cellinvasion of said substrate. Preferably the cancer cells are cultured inserum-free culture media so as to essentially avoid introducingcomplicating factors. In one embodiment, the invasion-inducing agent isa peptide, said peptide comprising the sequence PHSRN. In a preferredembodiment the invasion inducing agent is intact fibronectin.

[0011] While not limited to any mechanism, it is believed that cellsexposed to invasion-inducing agents in this manner are potentiallyrendered capable of invading the substrate. Indeed, the presentinvention contemplates stimulation of invasion by all cells of the body,including, but not limited to: epithelial (keratinocytes, mammary andprostate epithelial), connective tissue (fibroblasts), and muscle(myoblast) cells. Again, while not limited to any mechanism, it isbelieved that the invasion inducing agent comprising the sequence PHSRNbinds to the α5β1 receptor on the cancer cell and thereby inducesinvasion of the substrate. In this regard, the present inventionprovides a method of testing human cancer cells comprising: a)providing: i) a human cancer patient, ii) a fibronectin-free substrate,and iii) one or more invasion-inducing agents; b) obtaining α5β1integrin fibronectin receptor-expressing cancer cells from said patient;c) culturing said cells in serum-free culture media on said substrate inthe presence of said invasion-inducing agents; and d) detecting cancercell invasion of said substrate.

[0012] As noted above, the present invention also contemplates ascreening assay for identifying drugs that inhibit tumor invasion. Thepresent invention contemplates a screening assay utilizing the bindingactivity of fibronectin-derived peptides. In one embodiment, aninducible tumor cell line is placed in tissue culture on afibronectin-free substrate. Thereafter, as an inducible tumor cell line,the tumor will be induced (under ordinary conditions) by fibronectin orthe fibronectin-derived peptide to invade the substrate. However, inthis drug screening assay, candidate drug inhibitors are added to thetissue culture (this can be done individually or in mixtures). Where theinducible tumor cell is found to be inhibited from invading thesubstrate, a drug inhibitor is indicated.

[0013] It is not intended that the present invention be limited by thenature of the drugs screened in the screening assay of the presentinvention. A variety of drugs, including peptides, are contemplated.

[0014] Finally, the present invention contemplates chemotherapeutics fortreating invasive tumors. Specifically, a variety of anti-invasivechemotherapeutic agents are contemplated to antagonize theinvasion-promoting activity of the PHSRN peptide. In the preferredembodiment, the anti-invasive agent is a peptide with the amino acidsequence PHSCN. In another embodiment, the anti-invasive agent is apeptide which has an amino acid sequence comprising a sequence selectedfrom the group consisting of CHSRN, PCSRN, PHCRN, and PHSRC. In anotherembodiment, the anti-invasive agent is a peptide which has an amino acidsequence comprising PHSXN, where X is an amino acid selected from thegroup consisting of homo-cysteine, the D-isomer of cysteine, histidine,or penicillamine.

[0015] The present invention also contemplates an anti-invasive agentcomprising the amino acid sequence X₁HSX₂N, wherein X₁ is eitherproline, histidine, or not an amino acid, and X₂ is an amino acidselected from the group consisting of the L-isomer of cysteine, theD-isomer of cysteine, homo-cysteine, histidine, or penicillamine. Inanother embodiment, the present invention contemplates an anti-invasiveagent comprising the amino acid sequence X₁X₂X₃X₄X₅, wherein X₁ is anamino acid selected from the group consisting of proline, glycine,valine, histidine, isoleucine, phenylalanine, tyrosine, and tryptophan,and X₂ is an amino acid selected from the group consisting of histidine,proline, tyrosine, asparagine, glutamine, arginine, lysine,phenylalanine, and tryptophan, and X₃ is an amino acid selected from thegroup consisting of serine, threonine, alanine, tyrosine, leucine,histidine, asparagine, and glutamine, and X₄ is an amino acid selectedfrom the group consisting of cysteine, homo-cysteine, penicillamine,histidine, tyrosine, asparagine, glutamine, and methionine, and X₅ is anamino acid selected from the group consisting of asparagine, glutamine,serine, threonine, histidine, and tyrosine. In the preferred embodimentthe peptide is PHSCN, where the cysteine is the L-isomer.

[0016] It is further contemplated that the anti-invasive agents namedabove comprise the named amino acid sequence and additional amino acidsadded to the amino terminus, the carboxyl terminus, or both the aminoand carboxyl termini. In one embodiment, the anti-invasive agent is upto five hundred amino acids in length. It is also contemplated that, insome embodiments, the anti-invasive agents named above comprise apeptide with the amino terminus blocked by standard methods to preventdigestion by exopeptidases, for example by acetylation; and the carboxylterminus blocked by standard methods to prevent digestion byexopeptidases, for example, by amidation.

[0017] In this regard, the present invention provides a method oftreating cancer comprising: a) providing: i) a subject having cancer,and ii) a composition of matter comprising a peptide which inhibits thetumor invasion-promoting activity of the PHSRN sequence of plasmafibronectin; and b) administering said composition to said subject. Thepresent invention further contemplates using antagonists before and/orafter surgical removal of the primary tumor. In one embodiment, themethod comprises administering a PHSRN antagonist as adjunct therapywith additional chemotherapeutics.

[0018] While not limited to any mechanism, it is believed that theseanti-invasive chemotherapeutic agents antagonize the invasion-promotingactivity of the PHSRN sequence (e.g., of fibronectin) by blocking thebinding of this sequence to its receptor on tumor cells. Again, whilenot limited to any mechanism, it is believed that the PHSRN sequence maypromote invasion by acting to displace a divalent cation (Mg+2, Ca+2, orMn+) in the α5β1 receptor on metastatic tumor cells, and the above namedchemotherapeutic anti-invasive agents might act to inhibit this invasionby chelating one or more of these divalent cations.

[0019] In another embodiment, the present invention contemplatesanti-invasion antagonists to the IKVAV sequence of laminin, includingbut not limited to, peptides comprising the structure, ICVAV, andcorresponding peptide mimetics.

DESCRIPTION OF THE FIGURES

[0020]FIG. 1 schematically shows the one embodiment of the substrateused according to the present invention for testing tumor cells. Thespatial relationship of the ectoderm of the Strongylocentrotuspurpuratus embryo to its extracellular matrix and to blastocoelarstructures are shown (s, spicules; h, hyalin layer; c, ectoderm; b,subectodermal basement membrane; b1, blastocoel; g, stomach of theprimitive gut; c, coelomic pouches). The esophagus and intestine do notappear on the side of the embryo shown.

[0021]FIG. 2 is a graph showing the results of the testing of tumorcells on fibronectin-containing substrates and fibronectin-depletedsubstrates in vitro without the use of the invasion-inducing agents ofthe present invention.

[0022]FIG. 3 is a graph showing the results of the testing of tumorcells on fibronectin-depleted substrates in vitro with and withoutinvasion-inducing agents according one embodiment of the method of thepresent invention.

[0023]FIG. 4 is a graph showing the results of the testing of normalcells on fibronectin-depleted substrates in vitro with and withoutinvasion-inducing agents according one embodiment of the method of thepresent invention.

[0024]FIG. 5A is a graph showing the results of inhibiting serum-inducedhuman breast cancer cell invasion of the SU-ECM substrate with varyingconcentrations of the PHSCN peptide.

[0025]FIG. 5B is a graph showing the results of inhibiting PHSRN-inducedinvasion by both human breast cancer cells and normal human mammaryepithelial cells of the SU-ECM substrate with varying concentrations ofthe PHSCN peptide.

[0026]FIG. 6A is a graph showing the results of inhibiting serum-inducedhuman prostate cancer cell invasion of the SU-ECM substrate with varyingconcentrations of the PHSCN peptide.

[0027]FIG. 6B is a graph showing the results of inhibiting PHSRN-inducedinvasion by both human prostate cancer cells and normal prostateepithelial cells of the SU-ECM substrate with varying concentrations ofthe PHSCN peptide.

[0028]FIG. 7A is a graph showing the results of testing serum-inducedrat prostate cancer cell invasion of the SU-ECM substrate with andwithout the PHSCN peptide.

[0029]FIG. 7B is a graph showing the results of inhibiting PHSRN-inducedrat prostate cancer cell invasion of the SU-ECM substrate with varyingconcentrations of the PHSCN peptide.

[0030]FIG. 8 is a graph showing the results of inhibiting serum-inducedrat prostate cancer cell invasion of the SU-ECM substrate with varyingconcentrations of the PHS(homo)CN peptide.

[0031]FIG. 9A is a graph showing the results of testing tumor growth inrats injected with prostate cancer cells, with half of the ratsreceiving treatment with the PHSCN peptide, initiated in conjunctionwith the initial injection.

[0032]FIG. 9B is a graph showing the results of determining the meannumber of lung metastases in the two groups of rats described in FIG.9a.

[0033]FIG. 10A is a graph showing the results of testing tumor growth inrats injected with prostate cancer cells, with half of the ratsreceiving treatment with the PHSCN peptide, initiated 24 hours after theinitial cancer cell injection.

[0034]FIG. 10B is a graph showing the results of determining the meannumber of lung metastases in the two groups of rats described in FIG.10a.

[0035]FIG. 10C is a graph showing the results of determining the meanmass of intraperitoneal metastatic tissues in the two groups of ratsdescribed in FIG. 10a.

DEFINITIONS

[0036] The term “drug” as used herein, refers to any medicinal substanceused in humans or other animals. Encompassed within this definition arecompound analogs, naturally occurring, synthetic and recombinantpharmaceuticals, hormones, antimicrobials, neurotransmitters, etc.

[0037] The term “inducing agent” refers to any compound or moleculewhich is capable of causing (directly or indirectly) the invasion ofcells in a substrate. “Inducing agents” include, but are not limited to,PHSRN-containing peptides and related peptides (see below).

[0038] The term “receptors” refers to structures expressed by cells andwhich recognize binding molecules (e.g., ligands).

[0039] The term “antagonist” refers to molecules or compounds whichinhibit the action of a “native” or “natural” compound (such asfibronectin). Antagonists may or may not be homologous to these naturalcompounds in respect to conformation, charge or other characteristics.Thus, antagonists may be recognized by the same or different receptorsthat are recognized by the natural compound. “Antagonists” include, butare not limited to, PHSCN-containing peptides and related peptides (seebelow).

[0040] The term “host cell” or “cell” refers to any cell which is usedin any of the screening assays of the present invention. “Host cell” or“cell” also refers to any cell which either naturally expressesparticular receptors of interest or is genetically altered so as toproduce these normal or mutated receptors.

[0041] The term “chemotherapeutic agent” refers to molecules orcompounds which inhibit the growth or metastasis of tumors.“Chemotherapeutics” include, but are not limited to, PHSCN-containingpeptides and related peptides (see below).

[0042] As noted above, the present invention contemplates both the D andL isomers of cysteine which are identified collectively as “C”.

[0043] The present invention also contemplates homo-cysteine, which isidentified as “hC”.

DESCRIPTION OF THE INVENTION

[0044] The present invention generally relates to the treatment ofcancer, and more specifically, to the testing of cancer cells for theirability to invade tissues and cause metastases, and to theidentification and use of drugs to inhibit tumor invasion and growth. Asa prelude to metastasis, it is believed that cancer cellsproteolytically alter basement membranes underlying epithelia or theendothelial linings of blood and lymphatic vessels, invade through thedefects created by proteolysis, and enter the circulatory or lymphaticsystems to colonize distant sites. During this process, the secretion ofproteolytic enzymes is coupled with increased cellular motility andaltered adhesion. After their colonization of distant sites,metastasizing tumor cells proliferate to establish metastatic nodules.

[0045] As noted above, chemotherapeutic agents are currently employed toreduce the unrestricted growth of cancer cells, either prior to surgicalremoval of the tumor (neoadjuvant therapy) or after surgery (adjuvanttherapy). However, none of these methods has proved curative oncemetastasis has occurred. Since unrestricted invasive behavior is also ahallmark of metastatic tumor cells, methods for directly inhibitingtumor cell invasion and metastasis are needed.

[0046] A. Assays for Testing Tumor Invasion

[0047] Discovering how to inhibit the invasive behavior of tumor cellsto intervene in the metastatic cascade first requires the development ofassays with which to test tumor cell invasion in vitro. Two assaysystems are contemplated for use in the method of the present inventionto test the tumor cell invasion.

[0048] 1. Fibronectin-Depleted Substrates

[0049] In one assay system, the present invention contemplates usingfibronectin-depleted substrates. These are substrates that originallycontain fibronectin that are treated according to the methods of thepresent invention (see below) to remove fibronectin. It is not intendedthat the present invention be limited by the nature of the originalsubstrate; such fibronectin-containing substrates suitable for treatmentand depletion include: i) complex substrates containing a variety ofextracellular proteins and ii) less complex substrates containingfibronectin along with one or two other proteins (e.g., collagen,laminin, etc.).

[0050] It is also not intended that the present invention be limited bythe precise amount of fibronectin remaining after the substrate has beentreated. In other words, while the methods of the present inventionremove fibronectin, and in some embodiments, remove substantially allfibronectin, it is within the meaning of the term “fibronectin-depleted”substrate that a small amount of fibronectin remain in the substrate.

[0051] In one embodiment, the present invention contemplates using anextracellular matrix available commercially. For example, the presentinvention contemplates treating basement membrane matrices such as ECMGEL, a matrix from mouse sarcoma (commercially available from Sigma, St.Louis, Mo.). However, it is not intended that the present invention belimited by the particular fibronectin-containing substrate. For example,other commercially available substrates are contemplated, such as thecommonly used substrate Matrigel (available from Becton DickinsonLabware, Catalog #40234); Matrigel can be treated appropriatelyaccording to the methods of the present invention so as to render it“fibronectin-depleted” (see below). Untreated Matrigel (and similarsubstrates) have been used to demonstrate the importance of proteasesand motility factors in the invasion and metastasis of many tumors.However, these invasion substrates are not available as serum-freesubstrates; thus, the regulation of tumor cell invasive behavior byserum components, such as plasma fibronectin, is a complicating factorwith untreated Matrigel.

[0052] Consequently, the present invention contemplates afibronectin-free substrate. In this embodiment, Matrigel is treated sothat it is substantially fibronectin-free. The preparation offibronectin-free Matrigel involves “panning” the Matrigel substrate ongelatin as well as “panning” the substrate on anti-fibronectin antibody(anti-human fibronectin IgG is available commercially, such as antibodyfrom Promega Corporation, Madison, Wis.).

[0053] 2. Naturally Occurring Fibronectin-Free Substrates

[0054] In another embodiment, the present invention contemplatessubstrates that are naturally free of fibronectin; such a sourceprovides, for example, basement membranes permeable to select types ofnormally invasive cells, such membranes being naturally serum-free. Inone embodiment, the present invention contemplates sea urchins as asource of such membranes. In this regard, the ectoderm of sea urchinembryos is one cell thick, and secretes an underlying basement membrane(see FIG. 1) very similar to that of mammals. These embryos contain nocirculatory or lymphatic systems; and thus, their basement membranes areserum-free. In embryos, the subectodermal basement membrane functionssimultaneously as a migration substrate for several, specificmesenchymal cell types while it functions as an invasion substrate forothers. Sea urchin embryo basement membranes (SU-ECM) can be prepared bymild detergent treatment as described in D. Livant et al., CancerResearch 55:5085 (1995) and described in the Experimental section below.

[0055] Regardless of which of the two types of substrates are employed,the invasion substrates of the present invention are easy to prepare andgive rapid, highly consistent results with a variety of cells,including: a) cell lines from: i) primary and metastatic tumors, and ii)normal epithelial tissues; as well as b) cells from primary tissuesamples of both tumors, their surrounding normal tissues, and neonatalmelanocytes, fibroblasts, and keratinocytes from circumcised tissue.

[0056] In one embodiment, the present invention contemplates a method ofevaluating human cancer comprising: a) providing: i) a human cancerpatient (such as a patient with breast cancer or prostate cancer), ii) afibronectin-free substrate (for example, a fibronectin-depletedsubstrate) and iii) one or more invasion-inducing agents (discussedbelow); b) obtaining cancer cells from said patient (such as from abiopsy); c) contacting said cells ex vivo (i.e., outside the body) withsaid fibronectin-free substrate and said one or more invasion-inducingagents; and d) measuring the extent of cancer cell invasion of saidsubstrate. Preferably the cancer cells are cultured in serum-freeculture media so as to avoid introducing complicating factors.

[0057] 3. Inducing Agents

[0058] It is not intended that the present invention be limited by thenature of the agent that causes or induces cells to invade thefibronectin-free substrates of the present invention. Such agents can beidentified functionally by simply adding them to the cell culture andmeasuring the extent of invasion.

[0059] In one embodiment, the invasion-inducing agent comprises apeptide derived from fibronectin. In a preferred embodiment, theinvasion inducing agent is intact fibronectin.

[0060] While not limited to any mechanism, it is believed that cellsexposed to invasion-inducing agents in this manner are potentiallyrendered capable of invading the substrate. Again, while not limited toany mechanism, it is believed that the invasion inducing agentcomprising the sequence PHSRN binds to the α5β1 receptor on the cancercell and thereby induces invasion of the substrate. In this regard, thepresent invention provides a method of treating cells comprising: a)providing: i) cells expressing the α5β1 receptor, ii) a fibronectin-freesubstrate, and iii) one or more invasion-inducing agents; b) culturingsaid cells in serum-free culture media on said substrate in the presenceof said invasion-inducing agents; and d) measuring the extent of cellinvasion of said substrate. In one embodiment, the cells are normalepithelial cells or fibroblasts. In another embodiment, the cells arehuman cancer cells.

[0061] B. Drug Screening Assays

[0062] The present invention also contemplates a screening assay foridentifying drugs that inhibit tumor invasion. The present inventioncontemplates a screening assay (in the presence and absence of serum)utilizing the binding activity of fibronectin-derived peptides. In oneembodiment, an inducible tumor cell line is placed in tissue culture ona fibronectin-free substrate. The tumor cells will be induced (underordinary conditions) by the fibronectin-derived peptide to invade thesubstrate.

[0063] In one embodiment, the invasion-inducing agent comprises apeptide derived from fibronectin. In a preferred embodiment, saidpeptide comprises the sequence PHSRN. Of course, the peptide may belarger than five amino acids; indeed, the peptide fragment offibronectin may contain hundreds of additional residues (e.g., fivehundred amino acids). One such larger peptide is set forth in U.S. Pat.No. 5,492,890 (hereby incorporated by reference). In one embodiment, thePHSRN-containing peptide is less than one hundred amino acids in lengthand lacks the RGD sequence characteristic of fibronectin. A variety ofPHSRN-containing peptides are contemplated, including the PHSRN peptideitself and related peptides where additional amino acids are added tothe carboxyl terminus, including (but not limited to) peptidescomprising the sequence: 1) PHSRN, 2) PHSRNS, 3) PHSRNSI, 4) PHSRNSIT,5) PHSRNSITL, 6) PHSRNSITLT, 7) PHSRNSITLTN, 8) PHSRNSITLTNL, 9)PHSRNSITLTNLT, 10) PHSRNSITLTNLTP, and 11) PHSRNSITLTNLTPG.Alternatively, PHSRN-containing peptides are contemplated where aminoacids are added to the amino terminus, including (but not limited to)peptides comprising the sequence: 1) PEHFSGRPREDRVPHSRN, 2)EHFSGRPREDRVPHSRN, 3) HFSGRPREDRVPHSRN, 4) FSGRPREDRVPHSRN, 5)SGRPREDRVPHSRN, 6) GRPREDRVPHSRN, 7) RPREDRVPHSRN, 8) PREDRVPHSRN, 9)REDRVPHSRN, 10) EDRVPHSRN, 11) DRVPHSRN, 12) RVPHSRN, and 13) VPHSRN.Finally, the present invention contemplates PHSRN-containing peptideswhere amino acids are added to both the amino and carboxyl termini,including (but not limited to) peptides comprising the sequencePEHFSGRPREDRVPHSRNSITLTNLTPG, as well as peptides comprising portions orfragments of the PHSRN-containing sequence PEHFSGRPREDRVPHSRNSITLTNLTPG.

[0064] Peptides containing variations on the PHSRN motif arecontemplated. For example, the present invention also contemplatesPPSRN-containing peptides for use in the above-named assays. Suchpeptides may vary in length in the manner described above forPHSRN-containing peptides. Alternatively, PPSRN may be used as a peptideof five amino acids.

[0065] Similarly, peptides comprising the sequence -HHSRN-, -HPSRN-,-PHTRN-, -HHTRN-, -HPTRN-, -PHSNN-, -HHSNN-, -HPSNN-, -PHTNN-, -HHTNN-,-HPTNN-, -PHSKN-, -HHSKN-, -HPSKN-, -PHTKN-, -HHTKN-, -HPTKN-,-PHSRR-,-HHSRR-, -HPSRR-, -PHTRR-, -HHTRR-, -HPTRR-, -PHSNR-, -HHSNR-, -HPSNR-,-PHTNR-, -HHTNR-, -HPTNR-, -PHSKR-, -HHSKR-, -HPSKR-, -PHTKR-, -HHTKR-,-HPTKR-, -PHSRK-, -HHSRK-, -HPSRK-, -PHTRK-, -HHTRK-, -HPTRK-, -PHSNK-,-HHSNK-, -HPSNK-, -PHTNK-, -HHTNK-, -HPTNK-, -PHSKK-, -HHSKK-, -HPSKK-,-PHTKK-, -HHTKK-, or -HPTKK- are contemplated by the present invention.Such peptides can be used as five amino acid peptides or can be part ofa longer peptide (in the manner set forth above for PHSRN-containingpeptides).

[0066] In another embodiment, the present invention contemplates aninducing agent comprising the amino acid sequence X₁X₂X₃X₄X₅, wherein X₁is an amino acid selected from the group consisting of proline, glycine,valine, histidine, isoleucine, phenylalanine, tyrosine, and tryptophan,and X₂ is an amino acid selected from the group consisting of histidine,proline, tyrosine, asparagine, glutamine, arginine, lysine,phenylalanine, and tryptophan, and X₃ is an amino acid selected from thegroup consisting of serine, threonine, alanine, tyrosine, leucine,histidine, asparagine, and glutamine, and X₄ is an amino acid selectedfrom the group consisting of arginine, lysine, and histidine, and X₅ isan amino acid selected from the group consisting of asparagine,glutamine, serine, threonine, histidine, and tyrosine.

[0067] In this drug screening assay, candidate drug inhibitors are addedto the tissue culture (this can be done individually or in mixtures).Where the inducible tumor cell is found to be inhibited from invadingthe substrate, a drug inhibitor is indicated (see Examples section belowusing the PHSCN peptide).

[0068] It is not intended that the present invention be limited by thenature of the drugs screened in the screening assay of the presentinvention. A variety of drugs, including peptides and non-peptidemimetics, are contemplated.

[0069] It is also not intended that the present invention be limited bythe particular tumor cells used for drug testing. A variety of tumorcells (for both positive and negative controls) are contemplated(including but not limited to the cells set forth in Table 1 below).

[0070] C. Invasion-Inducing Agents and Antagonists

[0071] While an understanding of the mechanisms involved in metastaticcancer is not necessary to the successful practice of the presentinvention, it is believed that tumor cell invasion of basement membranesoccurs at several points in the metastatic cascade: (1) when epithelialtumor cells (such as those of breast and prostate cancers) leave theepithelium and enter the stroma, (2) when tumor cells enter thecirculatory or lymphatic systems, and (3) when tumor cells leave thecirculatory or lymphatic systems to invade distant sites. Thus,intervention in the induction of tumor cell invasiveness by using aPHSRN antagonist, such as the PHSCN peptide, to block tumor cellreceptors for this sequence is contemplated as a method for decreasingthe rate of metastasis.

[0072] One advantage of this strategy is that leukocytes are the onlynormal cells known to invade tissues to carry out their functions, andrelatively few leukocytes are invasive at a given time. Thus, relativelysmall doses of an anti-invasion antagonist which blocks the binding ofPHSRN to its receptor are required. Also, other than someimmunodepression, there should be relatively few side effects associatedwith anti-metastatic treatment using compounds designed to block theinduction of invasion. The lack of debilitating side effects expectedfrom anti-invasive therapy means that using it in combination withanti-proliferative agents would be uncomplicated, and that it could beused prior to surgery or even prophylactically to block tumor cellinvasion and metastasis.

[0073] The IKVAV sequence of laminin, a prevalent insoluble protein ofthe extracellular matrix, is known to stimulate liver colonization bymetastatic human colon cancer cells in athymic mice [see Bresalier etal., Cancer Research 55:2476 (1995)]. Since IKVAV, like PHSRN, containsa basic amino acid (K) which, by virtue of its positive charge, mightalso function to displace a divalent cation from its integrin receptorand stimulate invasion,the present invention contemplates applying thestrategy of developing anti-invasion antagonists to the IKVAV sequenceof laminin. TABLE 1 Designation And Origin Of Human Cell Lines AndStrains¹ Origin Cell Lines or Strains Colonic carcinoma SW1116, HCT116,SKCO-1, HT-29, KM12C, KM12SM, KM12L4, SW480 Pancreatic carcinoma BxPC-3,AsPC-1, Capan-2, MIA PaCa-2, Hs766T Colon adenoma VaCo 235 Lungcarcinoma A549 Prostate carcinoma PC-3, DU-145 Breast carcinoma 009P,013T, SUM-52 PE Lymphoma Daudi, Raji Breast epithelium 006FA Diploidfibroblast HCS (human corneal stroma), MRC-5

[0074] 1. Antagonists

[0075] It is not intended that the present invention be limited by thenature of the agent that inhibits tumor invasiveness. A variety ofanti-invasive chemotherapeutics are contemplated to antagonize theinvasion-promoting activity of the PHSRN sequence.

[0076] In the preferred embodiment, the anti-invasive agent is a peptidewith the amino acid sequence PHSCN. In another embodiment, theanti-invasive agent is a peptide which has an amino acid sequencecomprising a sequence selected from the group consisting of CHSRN,PCSRN, PHCRN, and PHSRC. In another embodiment, the anti-invasive agentis a peptide which has an amino acid sequence comprising PHSXN, where Xis an amino acid selected from the group consisting of homo-cysteine,the D-isomer of cysteine, histidine, or penicillamine.

[0077] The present invention also contemplates an anti-invasive agentcomprising the amino acid sequence X₁HSX₂N, wherein X₁ is eitherproline, histidine, or not an amino acid, and X₂ is an amino acidselected from the group consisting of the L-isomer of cysteine, theD-isomer of cysteine, homo-cysteine, histidine, or penicillamine. Inanother embodiment, the present invention contemplates an anti-invasiveagent comprising the amino acid sequence X₁X₂X₃X₄X₅, wherein X₁ is anamino acid selected from the group consisting of proline, glycine,valine, histidine, isoleucine, phenylalanine, tyrosine, and tryptophan,and X₂ is an amino acid selected from the group consisting of histidine,proline, tyrosine, asparagine, glutamine, arginine, lysine,phenylalanine, and tryptophan, and X₃ is an amino acid selected from thegroup consisting of serine, threonine, alanine, tyrosine, leucine,histidine, asparagine, and glutamine, and X₄ is an amino acid selectedfrom the group consisting of cysteine, homo-cysteine, penicillamine,histidine, tyrosine, asparagine, glutamine, and methionine, and X₅ is anamino acid selected from the group consisting of asparagine, glutamine,serine, threonine, histidine, and tyrosine. In the preferred embodimentthe peptide is PHSCN, where the cysteine is the L-isomer.

[0078] Similarly, peptides comprising the sequence -PSCN-, -HSCN-,-PSCN-, -HTCN-, -PTCN-, -HSCN-, -HSCN-, -PSCN-, -HTCN-, HTCN-, -PTCN-,-HSCN-, -HSCN-, -PSCN-, -HTCN-, -HTCN-, -PTCN-, -HSCR-, -HSCR-, -PSCR-,-HTCR-, -HTCR-, -PTCR-, -HSCR-, -HSCR-, -PSCR-, -HTCR-, -HTCR-, -PTCR-,-HSCR-, -HSCR-, -PSCR-, -HTCR-, -HTCR-, -PTCR-, -HSCK-, -HSCK-, -PSCK-,-HTCK-, -HTCK-, -PTCK-, -HSCK-, -HSCK-, -PSCK-, -HTCK-, -HTCK-, -PTCK-,-HSCK-, -HSCK-, -PSCK-, -HTCK-, -HTCK-, or -PTCK- are contemplated bythe present invention.

[0079] It is further contemplated that, in some embodiments, theanti-invasive agents named above comprise the named amino acid sequenceand additional amino acids added to the amino terminus, the carboxylterminus, or both the amino and carboxyl termini (in the manner setforth above for the PHSRN containing peptides, e.g., PHSRNSIT). In oneembodiment, the anti-invasive agent is up to five hundred amino acids inlength. It is also contemplated that, in some embodiments, theanti-invasive agents named above comprise a peptide with the aminoterminus blocked by standard methods to prevent digestion byexopeptidases, for example by acetylation; and the carboxyl terminusblocked by standard methods to prevent digestion by exopeptidases, forexample, by amidation.

[0080] In this regard, the present invention provides a method oftreating cancer comprising: a) providing: i) a subject having cancer,and ii) a composition of matter comprising a peptide, peptidederivative, or peptide mimetic which inhibits the tumorinvasion-promoting activity of a peptide comprising the amino acidsequence PHSRN, and b) administering said composition to said subject.The present invention further contemplates using antagonists beforeand/or after surgical removal of the primary tumor. In one embodiment,the method comprises administering a PHSRN antagonist as adjunct therapywith additional chemotherapeutics.

[0081] While not limited to any mechanism, it is believed that theseanti-invasive chemotherapeutic agents antagonize the invasion-promotingactivity of the PHSRN sequence (e.g., of fibronectin) by blocking thebinding of this sequence to its receptor on tumor cells. Again, whilenot limited to any mechanism, it is believed that the PHSRN sequence maypromote invasion by acting to displace a divalent cation (Mg+2, Ca+2, orMn+) in the α5β1 receptor on metastatic tumor cells, and the above namedchemotherapeutic anti-invasive agents might act to inhibit this invasionby chelating one or more of these divalent cations.

[0082] In another embodiment, the present invention contemplatesanti-invasion antagonists to the IKVAV sequence of laminin.

[0083] 2. Designing Mimetics

[0084] Compounds mimicking the necessary conformation for recognitionand docking to the receptor binding to the peptides of the presentinvention are contemplated as within the scope of this invention. Forexample, mimetics of PHSRN and PHSRN-antagonists are contemplated. Avariety of designs for such mimetics are possible. For example, cyclicPHSRN and PHSCN containing peptides, in which the necessary conformationfor binding is stabilized by nonpeptides, are specifically contemplated.U.S. Pat. No. 5,192,746 to Lobl, et al., U.S. Pat. No. 5,169,862 toBurke, Jr., et al., U.S. Pat. No. 5,539,085 to Bischoff, et al., U.S.Pat. No. 5,576,423 to Aversa, et al., U.S. Pat. No. 5,051,448 toShashoua, and U.S. Pat. No. 5,559,103 to Gaeta, et al., all herebyincorporated by reference, describe multiple methods for creating suchcompounds.

[0085] Synthesis of nonpeptide compounds that mimic peptide sequences isalso known in the art. Eldred, et al., (J. Med. Chem. 37:3882 (1994))describe nonpeptide antagonists that mimic the Arg-Gly-Asp sequence.Likewise, Ku, et al., (J. Med. Chem. 38:9 (1995)) give furtherelucidation of the synthesis of a series of such compounds. Suchnonpeptide compounds that mimic PHSRN and PHSRN-antagonists arespecifically contemplated by the present invention.

[0086] The present invention also contemplates synthetic mimickingcompounds that are multimeric compounds that repeat the relevant peptidesequences. In one embodiment of the present invention, it iscontemplated that the relevant peptide sequence is Pro-His-Ser-Arg-Asn;in another embodiment, the relevant peptide sequence isPro-His-Ser-Cys-Asn; in another embodiment, the relevant peptidesequence is Ile-Lys-Val-Ala-Val. As is known in the art, peptides can besynthesized by linking an amino group to a carboxyl group that has beenactivated by reaction with a coupling agent, such asdicyclohexylcarbodiimide (DCC). The attack of a free amino group on theactivated carboxyl leads to the formation of a peptide bond and therelease of dicyclohexylurea. It can be necessary to protect potentiallyreactive groups other than the amino and carboxyl groups intended toreact. For example, the amino group of the component containing theactivated carboxyl group can be blocked with a tertbutyloxycarbonylgroup. This protecting group can be subsequently removed by exposing thepeptide to dilute acid, which leaves peptide bonds intact. With thismethod, peptides can be readily synthesized by a solid phase method byadding amino acids stepwise to a growing peptide chain that is linked toan insoluble matrix, such as polystyrene beads. The carboxyl-terminalamino acid (with an amino protecting group) of the desired peptidesequence is first anchored to the polystyrene beads. The protectinggroup of the amino acid is then removed. The next amino acid (with theprotecting group) is added with the coupling agent. This is followed bya washing cycle. The cycle is repeated as necessary.

[0087] In one embodiment, the mimetics of the present invention arepeptides having sequence homology to the above-described PHSRN sequencesand PHSRN-antagonists. One common methodology for evaluating sequencehomology, and more importantly statistically significant similarities,is to use a Monte Carlo analysis using an algorithm written by Lipmanand Pearson to obtain a Z value. According to this analysis, a Z valuegreater than 6 indicates probable significance, and a Z value greaterthan 10 is considered to be statistically significant. W. R. Pearson andD. J. Lipman, Proc. Natl. Acad. Sci. (USA), 85:2444-2448 (1988); D. J.Lipman and W. R. Pearson, Science, 227:1435-1441 (1985). In the presentinvention, synthetic polypeptides useful in tumor therapy and inblocking invasion are those peptides with statistically significantsequence homology and similarity (Z value of Lipman and Pearsonalgorithm in Monte Carlo analysis exceeding 6).

[0088] 3. Antibody Inhibitors

[0089] The present invention contemplates all types of inhibitors oftumor invasion for use in both the assays and for therapeutic use. Inone embodiment, the present invention contemplates antibody inhibitors.The antibodies may be monoclonal or polyclonal, but polyclonalantibodies are often more effective inhibitors. It is within the scopeof this invention to include any second antibodies (monoclonal orpolyclonal) directed to the first antibodies discussed above. Both thefirst and second antibodies may be used in the detection assays or afirst antibody may be used with a commercially availableanti-immunoglobulin antibody. An antibody as contemplated hereinincludes any antibody specific to any region of a peptide involved inthe induction of tumor cell invasion. For example, the present inventioncontemplates antibodies reactive with PHSRN peptides (as well as therelated peptides set forth above).

[0090] Both polyclonal and monoclonal antibodies are obtainable byimmunization with peptides, as well as with enzymes or proteins, and alltypes are utilizable for immunoassays. The methods of obtaining bothtypes of sera are well known in the art. Polyclonal sera are lesspreferred but are relatively easily prepared by injection of a suitablelaboratory animal with an effective amount of the purified enzyme orprotein, or antigenic parts thereof, collecting serum from the animal,and isolating specific sera by any of the known immunoadsorbenttechniques. Although antibodies produced by this method are utilizablein virtually any type of immunoassay, they are generally less favoredbecause of the potential heterogeneity of the product.

[0091] The use of monoclonal antibodies in an immunoassay isparticularly preferred because of the ability to produce them in largequantities and the homogeneity of the product. The preparation ofhybridoma cell lines for monoclonal antibody production derived byfusing an immortal cell line and lymphocytes sensitized against theimmunogenic preparation can be done by techniques which are well knownto those who are skilled in the art. (See, for example Douillard andHoffman, Basic Facts about Hybridomas, in Compendium of Immunology VolII, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499,1975; European Journal of Immunology 6: 511-519, 1976).

[0092] Unlike preparation of polyclonal sera, the choice of animal isdependent on the availability of appropriate immortal lines capable offusing with lymphocytes. Mouse and rat have been the animals of choicein hybridoma technology and are preferably used. Humans can also beutilized as sources for sensitized lymphocytes if appropriateimmortalized human (or nonhuman) cell lines are available. For thepurpose of the present invention, the animal of choice may be injectedwith an antigenic amount, for example, from about 0.1 mg to about 20 mgof the enzyme or protein or antigenic parts thereof. Usually theinjecting material is emulsified in Freund's complete adjuvant. Boostinginjections may also be required. The detection of antibody productioncan be carried out by testing the antisera with appropriately labelledantigen. Lymphocytes can be obtained by removing the spleen of lymphnodes of sensitized animals in a sterile fashion and carrying outfusion. Alternatively, lymphocytes can be stimulated or immunized invitro, as described, for example, in Reading, Journal of ImmunologicalMethods 53: 261-291, 1982.

[0093] A number of cell lines suitable for fusion have been developedand the choice of any particular line for hybridization protocols isdirected by any one of a number of criteria such as speed, uniformity ofgrowth characteristics, deficiency of its metabolism for a component ofthe growth medium, and potential for good fusion frequency.

[0094] Intraspecies hybrids, particularly between like strains, workbetter than interspecies fusions. Several cell lines are available,including mutants selected for the loss of ability to secrete myelomaimmunoglobulin.

[0095] Cell fusion can be induced either by virus, such as Epstein-Barror Sendai virus, or polyethylene glycol. Polyethylene glycol (PEG) isthe most efficacious agent for the fusion of mammalian somatic cells.PEG itself may be toxic for cells and various concentrations should betested for effects on viability before attempting fusion. The molecularweight range of PEG may be varied from 1000 to 6000. It gives bestresults when diluted to from about 20% to about 70% (w/w) in saline orserum-free medium. Exposure to PEG at 37° C. for about 30 seconds ispreferred in the present case, utilizing murine cells. Extremes oftemperature (i.e., about 45° C.) are avoided, and preincubation of eachcomponent of the fusion system at 37° C. prior to fusion can be useful.The ratio between lymphocytes and malignant cells is optimized to avoidcell fusion among spleen cells and a range of from about 1:1 to about1:10 is commonly used.

[0096] The successfully fused cells can be separated from the myelomaline by any technique known by the art. The most common and preferredmethod is to choose a malignant line which is Hypoxthanine GuaninePhosphoribosyl Transferase (HGPRT) deficient, which will not grow in anaminopterin-containing medium used to allow only growth of hybrids andwhich is generally composed of hypoxthanine 1×10-4M, aminopterin1×10-5M, and thymidine 3×10-5M, commonly known as the HAT medium. Thefusion mixture can be grown in the HAT-containing culture mediumimmediately after the fusion 24 hours later. The feeding schedulesusually entail maintenance in HAT medium for two weeks and then feedingwith either regular culture medium or hypoxthanine, thymidine-containingmedium.

[0097] The growing colonies are then tested for the presence ofantibodies that recognize the antigenic preparation. Detection ofhybridoma antibodies can be performed using an assay where the antigenis bound to a solid support and allowed to react to hybridomasupernatants containing putative antibodies. The presence of antibodiesmay be detected by “sandwich” techniques using a variety of indicators.Most of the common methods are sufficiently sensitive for use in therange of antibody concentrations secreted during hybrid growth.

[0098] Cloning of hybrids can be carried out after 21-23 days of cellgrowth in selected medium. Cloning can be preformed by cell limitingdilution in fluid phase or by directly selecting single cells growing insemi-solid agarose. For limiting dilution, cell suspensions are dilutedserially to yield a statistical probability of having only one cell perwell. For the agarose technique, hybrids are seeded in a semi-solidupper layer, over a lower layer containing feeder cells. The coloniesfrom the upper layer may be picked up and eventually transferred towells.

[0099] Antibody-secreting hybrids can be grown in various tissue cultureflasks, yielding supernatants with variable concentrations ofantibodies. In order to obtain higher concentrations, hybrids may betransferred into animals to obtain inflammatory ascites.Antibody-containing ascites can be harvested 8-12 days afterintraperitoneal injection. The ascites contain a higher concentration ofantibodies but include both monoclonals and immunoglobulins from theinflammatory ascites. Antibody purification may then be achieved by, forexample, affinity chromatography.

[0100] A wide range of immunoassay techniques are available forevaluating the antibodies of the present invention as can be seen byreference to U.S. Pat. Nos. 4,016,043; 4,424,279 and 4,018,653, herebyincorporated by reference. This, of course, includes both single-siteand two-site, or “sandwich”, assays of the non-competitive types, aswell as in the traditional competitive binding assays.

[0101] 4. Administering Chemotherapeutics

[0102] It is contemplated that the antagonists of the present inventionbe administered systemically or locally to inhibit tumor cell invasionin cancer patients with locally advanced or metastatic cancers. They canbe administered intravenously, intrathecally, intraperitoneally as wellas orally. PHSRN antagonists (e.g., the PHSCN peptide), can beadministered alone or in combination with anti-proliferative drugs in aneoadjuvant setting to reduce the metastatic load in the patient priorto surgery; or they can be administered after surgery. Since PHSRNantagonists may depress wound healing (because the PHSRN sequence alsoelicits fibroblast invasion as described below), it may be necessary touse PHSRN antagonists some time after surgery to remove the tumor.

[0103] Since few cells in the body must invade in order to function,PHSRN antagonists administered systemically are not likely to cause thedebilitating side effects of cytotoxic chemotherapeutic agents. However,since they suppress invasion, they are likely to cause someimmunodepression. Even so, at the appropriate dosage, PSHRN antagonistsmay be administered prophylactically. In any case, it is contemplatedthat they may be administered in combination with cytotoxic agents. Thesimultaneous selection against the two fatal attributes of metastaticcells, unrestricted proliferation and invasion, is contemplated as avery powerful therapeutic strategy.

[0104] Where combinations are contemplated, it is not intended that thepresent invention be limited by the particular nature of thecombination. The present invention contemplates combinations as simplemixtures as well as chemical hybrids. An example of the latter is wherethe antagonist is covalently linked to a targeting carrier or to anactive pharmaceutical. Covalent binding can be accomplished by any oneof many commercially available crosslinking compounds.

[0105] It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided together with physiologically tolerableliquid, gel or solid carriers, diluents, adjuvants and excipients.

[0106] These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts.

[0107] Such compositions are typically prepared as liquid solutions orsuspensions, or in solid forms. Oral formulations for cancer usuallywill include such normally employed additives such as binders, fillers,carriers, preservatives, stabilizing agents, emulsifiers, buffers andexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, magnesiumcarbonate, and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%.

[0108] The compositions are also prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared.

[0109] The antagonists of the present invention are often mixed withdiluents or excipients which are physiological tolerable and compatible.Suitable diluents and excipients are, for example, water, saline,dextrose, glycerol, or the like, and combinations thereof. In addition,if desired the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing or pHbuffering agents.

[0110] Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases, suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides.

[0111] Anti-Thrombotics

[0112] In addition to using the PHSRN antagonists described above asanti-invasion chemotherapeutics, it is also contemplated that theseantagonists be used as anti-thrombotics. This use of the PHSRNantagonists described above is based on the discovery that PHSCNpeptide-treated blood appears in vivo to clot very slowly.

[0113] A number of anti-thrombotic agents are currently known whichinhibit clot formation by preventing platelet integrins from bindingfibrinogen or fibronectin. These anti-thrombotics, however, rely oncompetitive inhibition to prevent platelet integrins from binding tofibrinogen or fibronectin. In this maimer, large doses of these agentsare required to achieve the desired anti-thrombotic affect.

[0114] The present invention contemplates a more effective approachusing PHSRN-antagonists such as PHSCN. While the precise mechanism neednot be known to practice the invention it has been shown that theplatelet integrin, αIIbβ3, also binds the PHSRN sequence of plasmafibronectin. Thus, instead of utilizing competitive inhibition, thePHSRN-antagonists may directly inhibit platelet integrins from bindingfibronectin and aggregating. Specifically, the PHSCN peptide, or otherPHSRN-antagonists, may directly inhibit early stages in clot formationby binding to the αIIbβ3 receptors on platelets. This prevents plateletintegrins from binding fibronectin, a necessary part of plateletaggregation, thus inhibiting an integral step in the blood clottingcascade. In this manner, a comparatively small dose of the PHSCNpeptide, or other PHSRN antagonist, is contemplated as effectiveanti-thrombotic agents.

[0115] Wound Healing

[0116] As noted above, it is contemplated that PHSRN antagonists maydepress wound healing. This expectation is based on the discovery thatPHSRN-containing peptides promote wound healing.

[0117] In this regard, it should be noted that the therapy of wounds,particularly those which are made difficult to heal by disease, has beenattempted with a variety of purified growth factors or cytokines becausethese molecules can induce cellular proliferation or increase themotility of cells in wounds. Thus, if presented in the correct form andlocation at the right time, growth factors may greatly accelerate orenhance the healing of wounds by stimulating the growth of new tissue.Given the complexity and clinical variability of wounds, an obviousdifficulty with the application of specific, purified growth factors orcytokines to wounded tissue, alone or in combination, is that theirforms or specific distributions in the wound may not support theirnormal activities. Instead, the effectiveness of growth factors andcytokines in promoting the healing of wounded tissue may depend on theirsecretion by fibroblasts or macrophages.

[0118] The present invention contemplates a more effective approach;this approach involves methods that stimulate the invasion of the woundby the cells which synthesize the growth factors and cytokines active instimulating wound repair, especially monocytes, macrophages, andfibroblasts. This strategy allows the cells in their normal in vivosetting to secrete the active factors. This approach has a number ofadvantages: (1) the temporal and spatial distributions of the factorsare likely to be optimal because the normally active cells in theircorrect settings are secreting them; (2) all the appropriate factors arelikely to be present in their active forms, irrespective of whether theyhave been identified or cloned; (3) the sequential effects of thefactors in recruiting subsequent waves of cells involved in the healingprocess to the wound site are likely to be enhanced by the presence ofmore initiating cells in the wound.

[0119] The present invention is based on the discovery that the purePHSRN peptide or purified plasma fibronectin fragments containing it,and lacking the α4β1 integrin binding site in the IIICS region, aresufficient to stimulate fibroblast invasion of basement membranes invitro in the presence of serum or under serum-free conditions, whileintact plasma fibronectin fails to stimulate fibroblast invasion. PurePHSRN peptide has also been shown to stimulate keratinocyte invasion ofserum-free SU-ECM. Since, during wound reepithelialization,keratinocytes migrate through the connective tissue of the provisionalmatrix to “wall off” portions of the wound, as well as through theadjacent stroma, it is not surprising that they are also stimulated tomigrate through the matrix of SU-ECM invasion substrates by the PHSRNsequence. This suggests that this peptide, or proteinase-resistant formsof it, may have similar effects on fibroblasts, keratinocytes, andmonocytes/macrophages in vivo. Recruitment of fibroblasts ormonocytes/macrophages whose paracrine, regulatory effects on a varietyof neighboring cells are required for the early stages of wound healingis contemplated as a highly efficient and effective way to stimulate thecascade of regulatory interactions involved in wound healing becausethese cells will secrete the active factors or cytokines in the correcttemporal sequences and spatial locations to ensure their optimalactivities. Because it efficiently induces keratinocyte migrationthrough the extracellular matrix in vitro, the PHSRN peptide is alsolikely to stimulate wound reepithelialization directly. The use of thePHSRN peptide or structurally related molecules according to the presentinvention is to stimulate the entry of cells such as fibroblasts andmonocyte/macrophages into the provisional matrix of a wound, so that theentering cells themselves secrete the factors and cytokines active ininducing or potentiating wound healing. The use of the PHSRN peptide orstructurally related molecules is also intended to stimulate woundreepithelialization directly by inducing keratinocyte migration throughthe extracellular matrix.

[0120] Experimental

[0121] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0122] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); mM(millimolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); mAb (monoclonal antibody); MW (molecular weight);PBS (phophate buffered saline); U (units); d(days).

EXAMPLE 1 Production of Fibronectin-Free Substrates

[0123] This example describes a purification approach for removal ofplasma fibronectin (and/or cellular fibronectin) from a substrate(Matrigel). In this example, removal was attempted by affinitychromatography over Gelatin-Sepharose (a technique which can be used toremove plasma fibronectin from fetal calf serum).

[0124] The Gelatin-Sepharose beads were obtained from Pharmacia(Catalog#17-0956-01). Two Kontes columns were set up with about 2 mls ofGelatin-Sepharose beads at 4 C. to prevent gelling of the Matrigel. Thecolumns were then rinsed with about 10 column volumes of PBS to removethe preservative from the beads. The columns were drained to the top ofthe beads; then Matrigel was carefully added to the column. Once theMatrigel had entered the column, PBS was added to the top of the column.The Matrigel which was passed over the first column was collected andpassed over the second column. The fibronectin-depleted Matrigelcollected from the second column was plated on 48-well plates (150μl/well), sterilized under a UV light for 10 minutes and incubated at 37C. overnight. The Matrigel treated in this manner failed to form a gelat 37 C.

EXAMPLE 2 Production Of Fibronectin-Free Substrates

[0125] This example describes a purification approach for removal ofplasma fibronectin (and/or cellular fibronectin) from a substrate(Matrigel). In this example, removal was attempted by successive panningon gelatin. Eight wells of 24-well plate were coated with a 2% gelatinsolution (the gelatin was obtained from Becton Dickinson Labware,Catalog #11868). The wells were filled with the gelatin solution whichhad been heated to 50 C. and incubated for 3 minutes. Then the solutionwas removed and the wells were allowed to air dry. Following drying, thewells were thoroughly rinsed with ddH20 followed by two rinses with PBS.The plates were again allowed to dry; thereafter they were stored at −20C. until use. Matrigel was thawed on ice and then added to one of thewells of a gelatin-coated plate (between 800 μl and 1 ml of Matrigel wasadded to a well of a 24-well plate). The plate was placed in a bucket ofice in a 4 C. room on an orbital shaker where the Matrigel was incubatedin the well for two hours (although overnight incubation can be used).Following the incubation, the Matrigel was moved from the first well toa second well and then incubated for two hours under the sameconditions. This process was repeated until the Matrigel had beenincubated on all eight wells of the gelatin-coated plate.

[0126] Following the depletion of the Matrigel, it was collected inEppendorf tubes. It was then plated on a 48-well plate 150 μ1/well),sterilized under a UV light for 10 minutes and incubated at 37 C.overnight. The Matrigel formed as gel and the following day, cells wereadded to each well.

EXAMPLE 3 Production of Fibronectin-Free Substrates

[0127] This example describes a purification approach for removal ofplasma fibronectin (and/or cellular fibronectin) from a substrate(Matrigel). In this example, removal was attempted by gelatin panningfollowed by antibody panning.

[0128] Anti-fibronectin antibody-coated wells: Wells of a 24-well platewere coated with an anti-fibronectin antibody. A mouse monoclonalantibody to human fibronectin was obtained from Oncogene Science(Catalog #CP13). Each well was incubated with 1 ml of antibody at aconcentration of 30 μl/ml for 2 hours at room temperature. Each well wasthen incubated with a solution of 3% BSA in PBS for 2 hours at roomtemperature. Following the two incubation periods, the wells werethoroughly washed with PBS and stored at −20 C. until use.

[0129] Depleting Matrigel of Fibronectin: Matrigel was panned over eightgelatin-coated wells (as described above in Example 2) to remove most ofthe fibronectin and its fragments. Thereafter, the Matrigel was placedin the antibody-coated wells to remove any remaining fragments offibronectin which contain the cell-binding domain but not thegelatin-binding domain. The Matrigel was incubated in an ice bucket onan orbital shaker at 4 C. for 2 hours. Once the Matrigel was depleted,it was collected in Eppendorf tubes. The firbonectin-depleted Matrigelwas plated on a 48-well plate (150 μl/well), sterilized under a UV lightfor 10 minutes and incubated at 37 C. overnight. The Matrigel formed agel and the following day, cells were added to the wells.

EXAMPLE 4 Inducing Invasive Behavior of Tumor Cells

[0130] In this example, the role of plasma fibronectin in inducing theinvasive behaviors of metastatic breast and prostate cancer cells isdemonstrated. Human breast carcinoma cell lines SUM 52 PE and SUM 44 PEwere originally cultured from the pleural effusions of patients withmetastatic breast cancer; and SUM 102 was cultured from a primary,microinvasive breast carcinoma (Ethier, S. P., Mahack, M. L., Gullick,W. J., Frank, T. S., and Weber, B. L. Differential isolation of normalluminal mammary epithelial cells and breast cancer cells from primaryand metastatic sites using selective media. Cancer Res. 53: 627-635).The DU 145 metastatic human prostate cancer cell line was originallycultured from a brain metastasis (Stone, K. R., Mickey, D. D., Wunderli,H., Mickey, G. H., Paulsen, D. F. (1978) Isolation of a human prostatecarcinoma cell line (DU 145), Int. J. Cancer 21: 274-281. These cellsexpress α3β1 which has been shown to repress metalloproteinasetranscription upon binding the connecting segment of plasma Fn. Thesecell lines can all be cultured under serum-free conditions; thus theyare ideal for use in serum-free invasion assays on SU-ECM.

[0131] Adult Strongylocentrotus purpuratus sea urchins were obtainedfrom Pacific BioMarine, and their embryos were cultured to the earlypluteus stage in artificial sea water at 15° C. SU-ECM were preparedfrom them by treatment with nonionic detergent and sterilized bydilution in the appropriate media.

[0132] Cells were harvested by rinsing in Hanks' balanced salt solution,followed by brief treatment with 0.25% trypsin, 0.02% EDTA, andpelleting and resuspension in the appropriate medium with or without 5%FCS at a density of about 50,000 cells per ml. When appropriate,purified bovine plasma fibronectin (Sigma), purified 120 kDachymotryptic fragment (Gibco BRL), PHSRN or PHSCN peptides (synthesizedat the Biomedical Research Core Facilities of the University ofMichigan), or GRGDSP or GRGESP peptides (Gibco BRL) were added to theresuspended cells prior to placement of the cells on SU-ECM. In eachwell of a plate used for an invasion assay, SU-ECM were placed in 0.5 mlof the appropriate medium, and 0.5 ml of the resuspended cells droppedon their exterior surfaces. Invasion assays were incubated 1 to 16 hoursprior to assay. If some circumstances, invasion assays were fixed inphosphate-buffered saline (PBS) with 2% formaldehyde for 5 minutes atroom temperature, then rinsed into PBS.

[0133] Invasion assays were coded and scored blindly by microscopicexamination under phase contrast at 200- and 400-fold magnification.Each cell contacting an SU-ECM was scored for its position relative tothe exterior or interior surfaces. A cell was judged to have invaded ifit was located on an interior surface below the focal plane passingthrough the upper surface of the SU-ECM, but above the focal planepassing through its lower surface. The minimum viability of the cells ineach assay was always ascertained at the time of assay by determiningthe fraction of spread, adherent cells on the bottom of each wellscored.

[0134] An invasion frequency is defined as the fraction of cells incontact with basement membranes which were located in their interiors atthe time of assay. Thus, an invasion frequency of 1 denotes invasion by100% of the cells in contact with basement membranes. Invasionfrequencies were determined multiple times for each cell type assayed.For each type of cell assayed the mean and standard deviation of theinvasion frequencies were calculated.

[0135] The invasion-inducing sequences of plasma fibronectin were mappedto a peptide sequence 5 amino acids long, the PHSRN peptide, for bothmetastatic breast and prostate cancer cells. Since the PHSRN sequence ispresent in plasma fibronectin, a significant component of serum,eliciting the regulatory role of this sequence was only possible becauseof the availability of a serum-free in vitro invasion substrate. Itshould be noted that neonatal, human fibroblasts are also induced withthe PHSRN peptide to invade serum-free SU-ECM. Although fibroblasts donot invade SU-ECM in the presence of serum, the 120 kDa fragment ofplasma fibronectin containing the PHSRN sequence can induce fibroblastinvasion equally well in the presence of serum or in its absence.

[0136] When taken together, the results of experiments showing that thePHSRN sequence of plasma fibronectin induces the invasive behaviors ofboth metastatic breast and prostate cancer cells, as well as that ofnormal fibroblasts suggest the intriguing possibility that the invasivebehavior associated with tumor cell metastasis may result from defectsin the regulation of the normal invasive behaviors associated with woundhealing.

EXAMPLE 5 Testing Tumor Cells on Fibronectin-Depleted Substrates

[0137] This example describes an approach to test cancer cells in vitroon substrates with and without invasion-inducing agents. The depletedpreparation of Matrigel (see Example 2, above) and untreated Matrigelwere used to test DU-145 metastatic prostate cancer cells. When platedon the depleted medium, the cancer cells failed to invade the matrix(see FIG. 2). Indeed, it was evident that these cells were sitting onthe surface of the depleted Matrigel because the Matrigel surface wasslightly tilted; this was visible through the microscope as a gradualprogressive, uniform change in the focal plane for the monolayer ofDU-145 cells.

[0138] The addition of 0.5 μl/ml of the PHSRN peptide to the depletedMatrigel was sufficient to restore the full DU-145 invasiveness (seeFIG. 3). Clearly, gelatin panning removes fibronectin such that cancercells are unable to invade. Since the addition of PHSRN peptide insolution fully restores the DU-145 invasive phenotype, blocking theeffect of PHSRN is an effective strategy for therapeutic intervention intumor cell invasion and metastasis.

EXAMPLE 6 Improving Gelatin Depletion as Measured by FibroblastInvasiveness

[0139] In this example, normal, neonatal fibroblasts were tested on thedepleted Matrigel material prepared according to Example 3 above (i.e.,antibody depletion). As shown in FIG. 4, panning with an antibody aftergelatin depletion improved the method for removal, as measured by thereduced invasiveness of fibroblasts. On the other hand, invasiveness ofthe fibroblasts could be induced by the addition of the PHSRN peptide.

[0140] The success of antibody panning suggests the feasibility ofremoving other components by the antibody panning methods. Other serumcomponents, such as thrombospondin, growth factors and cytokines arecontemplated by the present invention for removal by the appropriate(commercially available) antibody.

EXAMPLE 7 Conjugation of PHSRN-Containing Peptides

[0141] In this example, the preparation of a peptide conjugate isdescribed. The synthetic peptide NH₂-PHSRNC can be prepared commercially(e.g., Multiple Peptide Systems, San Diego, Calif.). The cysteine isadded to facilitate conjugation to other proteins.

[0142] In order to prepare a protein for conjugation (e.g., BSA), it isdissolved in buffer (e.g., 0.01 M NaPO₄, pH 7.0) to a finalconcentration of approximately 20 mg/ml. At the same timen-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS” available fromPierce) is dissolved in N,N-dimethyl formamide to a concentration of 5mg/ml. The MBS solution, 0.51 ml, is added to 3.25 ml of the proteinsolution and incubated for 30 minutes at room temperature with stirringevery 5 minutes. The MBS-activated protein is then purified bychromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bed volume)equilibrated with 50 mM NaPO₄, pH 7.0 buffer. Peak fractions are pooled(6.0 ml).

[0143] The above-described cysteine-modified peptide (20 mg) is added tothe activated protein mixture, stirred until the peptide is dissolvedand incubated 3 hours at room temperature. Within 20 minutes, thereaction mixture becomes cloudy and precipitates form. After 3 hours,the reaction mixture is centrifuged at 10,000×g for 10 min and thesupernatant analyzed for protein content. The conjugate precipitate iswashed three times with PBS and stored at 4° C.

[0144] From the above, it should be clear that the present inventionprovides a method of testing a wide variety of tumor types, and inparticular identifying invasive tumors. With a means of identifying suchtumors (now provided by the present invention) and distinguishing suchtumors from non-invasive cancer, the physician is able to change and/oroptimize therapy. Importantly, the antagonists of the present invention(and other drugs developed by use of the screening assay of the presentinvention) will provide treatment directed an invasive cells (andtherefore associated with minimal host toxicity).

EXAMPLE 8 Inhibiting Invasion of Human Breast Cancer Cells

[0145] In this example, the role of the PHSCN peptide in inhibiting theinvasive behavior of metastatic breast cancer cells is demonstrated. Themethod of Example 4 is employed, with the addition of varyingconcentrations of the PHSCN peptide.

[0146] Example 4 indicates that SUM-52 cells (in medium with 5% fecalcalf serum) are induced to invade the SU-ECM substrate in the presenceof serum fibronectin or just the PHSRN sequence of fibronectin. Thus,the procedure in Example 4 provides a method for determining theinhibitory potential of the PHSCN peptide by comparing the number ofcell invasions in the presence of the PHSCN peptide, with the number ofcell invasions in the absence of the PHSCN peptide.

[0147] The results of adding varying concentrations of the PHSCN peptideto serum-induced metastatic SUM-52 PE breast cancer cells are presentedin FIG. 5A. The logs of the PHSCN peptide concentrations in ng per mlare plotted on the X axis. The percentages of invaded SUM 52 PE cellsrelative to the percentage invaded in the absence of the PHSCN peptideare plotted on the Y axis. Mean invasion percentages are shown withtheir first standard deviations. Clearly, the PHSCN peptide exhibits asubstantial inhibitory affect on these cells, even at relatively lowconcentrations. The PHSCN peptide's inhibitory affect is furtherdemonstrated by the fact that relatively high concentrations causecomplete inhibition.

[0148] The results of adding varying concentrations of the PHSCN peptideto PHSRN-induced metastatic SUM-52 PE breast cancer cells (in serum freemedium) and normal human mammary epithelial cells (in 10% FCS), arepresented in FIG. 5B. All invasion assays were carried out in 100 ng perml of the PHSRN peptide to induce invasion. Again, the PHSCN peptideexhibits a substantial inhibitory affect on both cell lines at lowconcentrations, and almost complete inhibition at higher concentrations.

[0149] This example demonstrates the PHSCN peptide is an effectiveinhibitor of human breast cancer cell invasion. In this manner, thePHSCN peptide, or related sequences, are likely to provide effectivetherapy for human breast cancer by preventing the lethal affects oftumor cell metastasis.

EXAMPLE 9 Inhibiting Invasion of Human Prostate Cancer Cells

[0150] In this example, the role of the PHSCN peptide in inhibiting theinvasive behavior of metastatic prostate cancer cells is demonstrated.The method of Example 4 is employed, with the addition of varyingconcentrations of the PHSCN peptide.

[0151] Example 4 indicates that DU-145 cells are induced to invade theSU-ECM substrate in the presence of serum fibronectin or just the PHSRNsequence of fibronectin. Thus, the procedure in Example 4 provides amethod for determining the inhibitory potential of the PHSCN peptide bycomparing the number of cell invasions in the presence of the PHSCNpeptide, with the number of cell invasions in the absence of the PHSCNpeptide.

[0152] The results of adding varying concentrations of the PHSCN peptideto serum-induced invasion of metastatic DU-145 prostate cancer cells (in10% serum) are presented in FIG. 6A. The logs of the PHSCNconcentrations are plotted on the X axis. The percentages of invadedDU-145 cells relative to the percentage invaded in the absence of thePHSCN peptide are plotted on the Y axis. Mean invasion percentages areshown with their first standard deviations. Clearly, the PHSCN peptideexhibits a substantial inhibitory affect on these cells, even atrelatively low concentrations. The PHSCN peptide's inhibitory affect isfurther demonstrated by the fact that relatively high concentrationscause complete inhibition.

[0153] The results of adding varying concentrations of the PHSCN peptideto PHSRN-induced metastatic DU-145 prostate cancer cells (in serum-freemedium) and normal human prostate epithelial cells (in 10% FCS), arepresented in FIG. 6B. All invasion assays were carried out in 100 ng perml of the PHSRN peptide to induce invasion. Again, the results show thatthe PHSCN peptide exhibits a substantial inhibitory affect on both celllines at low concentrations, and almost complete inhibition at higherconcentrations.

[0154] This example demonstrates the PHSCN peptide is an effectiveinhibitor of human prostate cancer cell invasion. In this manner, thePHSCN peptide may provide an effective therapy for human prostate cancerby preventing the lethal affects of tumor cell metastasis.

EXAMPLE 10 Inhibiting Invasion of Rat Prostate Cancer Cells

[0155] In this example, the role of the PHSCN peptide in inhibiting theinvasive behavior of rat metastatic prostate carcinoma MatLyLu (MLL)cells is demonstrated (see Example 4 for the general procedureemployed). The result of adding 1 microgram per ml of the PHSCN peptideto serum-induced MLL cells causes complete inhibition of invasion (seeFIG. 7A).

[0156] The result of adding a varying concentration of the PHSCN peptideto PHSRN-induced MLL cells in serum free media is shown in FIG. 7B,where 100 ng per ml of PHSRN was used to induce invasion. FIG. 7Bindicates that the PHSCN peptide exhibits a substantial inhibitoryaffect even at low concentrations, and almost complete inhibition athigher concentrations. This example demonstrates invasion of ratprostate cancer cells is inhibited in the same manner as human breastcancer cells (see Example 8) and human prostate cancer cells (seeExample 9).

EXAMPLE 11 Inhibiting Invasion of Rat Prostate Cancer Cells

[0157] In this example, the role of a homo-cysteine containing peptide(i.e., PHS(hC)N) in inhibiting the invasive behavior of rat metastaticprostate carcinoma MatLyLu (MLL) cells is demonstrated. The proceduredescribed in Example 10, was employed using SU-ECM substrates in 10% FCSand PHS(hC)N instead of PHSCN. The result of adding varyingconcentrations of the PHS(hC)N peptide to serum-induced MLL cellsindicates this peptide also has an inhibitory affect on cell invasion(see FIG. 8). As with the PHSCN peptide, the PHS(hC)N peptidesubstantially inhibits invasion at lower concentrations, and completelyinhibits invasion at higher concentrations. This example demonstratesthat the PHS(hC)N peptide has a similar inhibitory affect as the PHSCNpeptide.

EXAMPLE 12 Inhibiting Growth and Metastasis of Prostate Cancer Tumors inVivo

[0158] In this example, the role of the PHSCN peptide in inhibiting thegrowth and metastasis of prostate cancer tumors in vivo is demonstrated.In the first part of this example, four Copenhagen rats were injectedwith 500,000 MatLyLu (MLL) cells subcutaneously in the thigh. Two ofthese rats also received 1 mg of the PHSCN peptide along with theinjected MLL cells, and thereafter received 1 mg of the PHSCN peptideinjected in their tail vein three time per week for two weeks. The othertwo injected rats were left untreated. Tumor sizes were measured withcalipers on day 14, and the tumors in the untreated rats were removed.The results depicted in FIG. 9A, clearly demonstrate that the PHSCNpeptide significantly slows the growth of injected MLL tumors in rats.It is possible that the ability of the PHSCN peptide to slow tumorgrowth is due to its inhibition of tumor invasion by normal endothelialcells, an anti-angiogenic effect.

[0159] Two weeks after the size of the tumors were measured, the ratswere sacrificed and the mean number of lung metastases was determined at10-fold magnification. The mean number of lung metastases in theuntreated mice (MLL only) was nearly 35 in spite of the fact that theinitial prostate tumors had been removed when their size was measured.The mean number of lung metastases in the treated mice (MLL+PHSCN) wasless than 5, even though the initial prostate tumors were never removedbecause they were too small. This striking difference in mean number ofmetastases, depicted in FIG. 9B, indicates that the PHSCN peptidesignificantly inhibits tumor cell metastasis in rats. In this manner,the PHSCN peptide provides effective in vivo therapy for cancer bypreventing the lethal effects of tumor cell growth and metastasis.

EXAMPLE 13 Inhibiting Growth and Metastasis of Prostate Cancer in Vivo

[0160] In this example, as in Example 12, the role of the PHSCN peptidein inhibiting the growth and metastasis of prostate cancer tumors invivo is demonstrated. In the first part of this example, 20 Copenhagenrats were injected with 500,000 MatLyuLu (MLL) cells subcutaneously inthe thigh. To more closely approximate a real clinical situation, PHSCNpeptide treatment of 10 of these rats was initiated after 24 hours,instead of immediately. The 10 treated rats (MLL/PHSCN) received a totalof 5 i.v. injections of 1 mg of the PHSCN peptide through the tail veinover two weeks. Tumor sizes were measured with calipers on day 14, andthe tumors in the untreated rats were removed. Since the injected tumorsin the MLL/PHSCN rats were still small, they were retained in the ratsfor another 7 to 9 days following the last PHSCN injection. At thistime, their sizes were all greater than 2 cm, and they were alsoremoved. The result of the first part of this example, depicted in FIG.10A, clearly indicates that the PHSCN peptide, even when administeredafter the tumor cells have “seeded”, substantially slows the growth ofrat prostate cancer tumors.

[0161] The dramatic growth-inhibitory effect of the PHSCN peptide on MLLtumors may be due to their inhibition of the invasion of hostendothelial cells into the tumor. Host endothelial cell invasion may beinduced by the secretion of large amounts of proteinases from thetumors, and the resulting fragmentation of host plasma fibronectin.Fibronectin fragments have been shown to stimulate themigratory/invasive behaviors of normal mesenchymal and endothelialcells. This angiogenic process is believed to occur during normal woundhealing. Thus, the ability of metastatic cells to be constitutivelyinduced by intact plasma fibronectin to express proteinases and invademay play a central role both in tumor cell invasion and in tumor growth.In this manner, the PHSCN peptide is an effective chemotherapeutic toprevent the growth of tumors in vivo.

[0162] In the second part of this example, the MLL/PHSCN rats received 2more i.v. doses of the PHSCN peptide prior to sacrifice. Ten days afterthe sizes of the injected primary tumors were determined, all the ratsin the two groups (MLL only and MLL/PHSCN) were sacrificed, and thenumber of lung metastases was determined at 7.5-fold magnification. Ascan be seen in FIG. 10B, there is a significant reduction in the meannumbers of lung metastases in the rats which received PHSCN treatment ascompared to the untreated rats.

[0163] The 20 rats described in parts one and two of this example werealso examined for metastatic tissues in their lymphatic systems. All ofthese metastases were dissected and weighed. FIG. 10C plots the meanmasses of intraperitoneal metastases (grams) for the two groups of 10rats. As is clearly demonstrated, there is a significant reduction inthe mean masses of lymphatic metastases in the rats which received PHSCNpeptide treatment, as compared to the untreated rats. This may be due tothe anti-angiogenic effect of the PHSCN peptide, as described in partone of this example. In this manner, the PHSCN peptide maybe aneffective anti-metastatic, growth-inhibiting chemotherapeutic agent foruse in the treatment of cancer.

[0164] From the above, it should be clear that the present inventionprovides an anticancer approach that is reliable for a wide variety oftumor types, and particularly suitable for invasive tumors. Importantly,the treatment is effective with minimal host toxicity.

1. A composition of matter comprising a peptide which inhibits the tumorinvasion-promoting activity of a peptide comprising the amino acidsequence PHSRN.
 2. The composition of claim 1, wherein said peptide isup to five hundred amino acids in length.
 3. The composition of claim 1,wherein said peptide has an amino acid sequence comprising PHSCN.
 4. Thecomposition, as claimed in claim 3, wherein said peptide containsadditional amino acids added to the amino terminus, the carboxylterminus, or both the amino and carboxyl termini.
 5. The composition, asclaimed in claim 1, wherein said first peptide is a cyclicPHSCN-containing peptide.
 6. The composition of claim 1, wherein saidpeptide has an amino acid sequence comprising a sequence selected fromthe group consisting of CHSRN, PCSRN, PHCRN, PHSRC.
 7. The peptide ofclaim 6, wherein said peptide contains additional amino acids added tothe amino terminus, the carboxyl terminus, or both the amino andcarboxyl termini.
 8. A non-peptide composition of matter which inhibitsthe tumor invasion-promoting activity of a peptide comprising the aminoacid sequence PHSRN.
 9. A first peptide which inhibits the tumorinvasion-promoting activity of a second peptide, wherein said firstpeptide's amino terminus is blocked with an acetyl group, and itscarboxyl terminus is blocked with an amide group, and said secondpeptide comprises the amino acid sequence PHSRN.
 10. The first peptideof claim 9, wherein said peptide is up to five hundred amino acids inlength.
 11. The first peptide of claim 9, wherein said first peptide hasan amino acid sequence comprising PHSCN.
 12. The first peptide of claim11, wherein said peptide contains additional amino acids added to theamino terminus, the carboxyl terminus, or both the amino and carboxyltermini.
 13. The first peptide of claim 9, wherein said first peptidehas an amino acid sequence comprising a sequence selected from the groupconsisting of CHSRN, PCSRN, PHCRN, PHSRC.
 14. The peptide, as claimed inclaim 13, wherein said peptide contains additional amino acids added tothe amino terminus, the carboxyl terminus, or both the amino andcarboxyl termini.